Mammalian tumor susceptibility gene products and their uses

ABSTRACT

The present invention provides methods and compositions for regulating ubiquitination in a cell. In particular, the present invention provides purified polypeptides comprising an ubiquitination-regulating domain. The invention also provides methods of using such polypeptides for screening for agents, for producing antibodies, and for treatment of diseases, e.g., proliferative diseases, neurodegenerative diseases, autoimmune diseases, metabolic disease and developmental abnormalities. The invention further provides antibodies that bind an ubiquitination-regulating domain and agents and antibodies that regulate ubiquitination in cells, e.g., by modulating the interaction between a TSG101 protein and an MDM2 protein.

FIELD OF THE INVENTION

The invention relates to protein ubiquitination, and to mammalian cellproliferation.

BACKGROUND OF THE INVENTION

The TSG101 tumor susceptibility gene initially was identified by thereversible neoplasia associated with deficiency of its protein productin mouse fibroblasts (1). Deficiency of TSG101 induced by antisense RNAin NIH3T3 cells leads to colony formation in 0.5% agar, focus formationin monolayer cell cultures, and the ability to form metastatic tumors inathymic nude mice (1). Turn off of TSG101-inactivating antisense RNAreverses these features of neoplastic transformation as well as thenuclear, microtubule, and mitotic spindle abnormalities observed inTSG101-deficient cells (1, 2). The steady-state level of TSG101 proteinnormally is regulated post-translationally in cells within a narrowrange (3), and overexpression of TSG101 from an adventitious promotercan also lead to cell cycling abnormalities (2) and neoplastictransformation (1). Truncated TSG101 transcripts, which are observed ina variety of human tumors as well as in normal cells (4-7), have beenattributed to aberrant or alternative RNA splicing (8) and have beencorrelated with both cellular stress (4, 5) and mutation of p53 (5). TheTSG101 protein contains motifs common to transcription regulators (1)and can modulate transcriptional activation by steroid hormone receptors(9-11).

Sequence analysis has also suggested that TSG101, which is expressed inmammalian cells from the earliest stages of embryonic development and inmultiple tissues of adult mice (8), may additionally have a role in theregulation of ubiquitin-mediated proteolysis (12, 13). The N-terminalregion of the TSG101 protein contains a domain (Ubc) that resembles thecatalytically active region of ubiquitin conjugases (E2 enzymes) butlacks an active site cysteine residue crucial to the function of theseenzymes (12-14), leading to speculation that TSG101 may act as adominant negative inhibitor of ubiquitination (12, 13).

p53 is a key tumor suppressor that transcriptionally activates MDM2 aswell as other genes implicated in both cell growth and cell death(15-18). MDM2 in turn negatively regulates p53 by promoting itsubiquitin-mediated degradation (19-21). Despite p53/MDM2 feedbackcontrol, p53 accumulates in cells soon after DNA damage, hypoxia, andother types of stress, suggesting that the actions of MDM2 and p53 oneach other are themselves regulated (16). Several mechanisms for suchregulation have been proposed (16, 17) and recent evidence indicatesthat alteration of MDM2 stability mediated by its interactions withother cellular proteins may have a role in this process (22-24).

There is a continuing need in the art for new and better methods ofmodulating proliferation of mammalian cells. The present inventionaddresses this need.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions for regulatingubiquitination in a cell. In particular, the present invention providespurified polypeptides comprising an ubiquitination-regulating domain.The invention also provides methods of using such polypeptides forscreening for agents, for producing antibodies, and for treatment ofdiseases, e.g., proliferative diseases, neurodegenerative diseases,autoimmune diseases, and developmental abnormalities. The inventionfurther provides antibodies that bind an ubiquitination-regulatingdomain and agents and antibodies that regulate ubiquitination in cells,e.g., by modulating the interaction between a TSG101 protein and an MDM2protein.

In one embodiment, the present invention provides antibodies that bindspecifically to a polypeptide comprising an ubiquitination-regulatingdomain. In a preferred embodiment, the invention provides antibodiesthat bind to the ubiquitination-regulating domain, or a functionalfragment thereof, of a TSG101 protein, e.g., a human TSG101 protein. Inanother preferred embodiment, the invention provides antibodies thatbind to an ubiquitination-regulating domain comprising amino acidresidues 1-140 of a human TSG101 protein, e.g., amino acid residues1-140 of SEQ ID NO:1. In still another preferred embodiment, theubiquitination-regulating domain comprises amino acid residues 50-140 ofa human TSG101, e.g., amino acid residues 50-140 of SEQ ID NO:1. Instill another preferred embodiment, the invention provides antibodiesthat bind to an ubiquitination-regulating domain comprising amino acidresidues 140-250 of a human TSG101 protein, e.g., amino acid residues140-250 of SEQ ID NO:1. In still other embodiments, theubiquitination-regulating domain may comprises, e.g., amino acidresidues 10-140, 20-140, 30-140, 40-140, 1-160, 1-180, 1-200, 1-220,50-250 or 1-250 of a human TSG101, e.g., amino acid residues 10-140,20-140, 30-140, 40-140, 1-160, 1-180, 1-200, 1-220, 50-250 or 1-250 ofSEQ ID NO:1. The present invention also provides methods of producingsuch antibodies that binds specifically to an ubiquitination-regulatingdomain. In the methods of the invention, antibodies are raised against apolypeptide comprising the ubiquitination-regulating domain. Anypolypeptide that comprises an ubiquitination-regulating domain can beused to produce the antibodies of the invention.

In other embodiments, the present invention provides antibodies thatbind a TSG101 protein at a domain other than the Ub domain, e.g., at thecoiled-coil domain, or the steady box domain such that the bindingmodulates the function of the ubiquitination-regulating domain.

The present invention also provides a cell comprising a polynucleotideencoding an ubiquitination-regulating domain operationally linked to aregulatory sequence such that the cell expresses theubiquitination-regulating domain. The invention also provides a cellcomprising (i) a polynucleotide encoding an ubiquitination-regulatingdomain operationally linked to a regulatory sequence; and (ii) apolynucleotide encoding MDM2 protein operationally linked to aregulatory sequence, such that the cell expresses theubiquitination-regulating domain and the MDM2 protein. The inventionalso provides a cell comprising (i) a polynucleotide encoding anubiquitination-regulating domain operationally linked to a regulatorysequence; (ii) a polynucleotide encoding MDM2 protein operationallylinked to a regulatory sequence; and (iii) a polynucleotide encoding p53protein operationally linked to a regulatory sequence, such that thecell expresses the ubiquitination-regulating domain, the MDM2 protein,and the p53 protein. The ubiquitination-regulating domain can be anubiquitination-regulating domain, or a functional fragment thereof, of aTSG101 protein, e.g., a human TSG101 protein. In a preferred embodiment,the ubiquitination-regulating domain comprises amino acid residues 1-140of a human TSG101 protein, e.g., amino acid residues 1-140 of SEQ IDNO:1. In another preferred embodiment, the ubiquitination-regulatingdomain comprises amino acid residues 50-140 of a human TSG101, e.g.,amino acid residues 50-140 of SEQ ID NO:1. In still another preferredembodiment, the ubiquitination-regulating domain comprises amino acidresidues 140-250 of a human TSG101 protein, e.g., amino acid residues140-250 of SEQ ID NO:1. In still other embodiments, theubiquitination-regulating domain may comprises amino acid residues10-140, 20-140, 30-140, 40-140, 1-160, 1-180, 1-200, 1-220, 50-250 or1-250 of a human TSG101, e.g., amino acid residues 10-140, 20-140,30-140, 40-140, 1-160, 1-180, 1-200, 1-220, 50-250 or 1-250 of SEQ IDNO:1.

The invention provides methods of identifying an agent that modulatesthe interaction of a TSG101 protein with an MDM2 protein. The methodscomprise screening candidate agents using a screening assay comprising acell expressing MDM2 and a polypeptide comprising anubiquitination-regulating domain, or a functional fragment thereof, ofthe TSG101 protein. In a specific embodiment, the invention provides amethod of identifying an agent that is capable of modulating theinteraction of a TSG101 protein with MDM2, comprising: (a) contacting acell expressing MDM2 and a polypeptide comprising anubiquitination-regulating domain, or a functional fragment thereof, ofthe TSG101 protein with the agent and measuring MDM2 level in the cell;(b) contacting a cell expressing MDM2 but not anubiquitination-regulating domain, or a functional fragment thereof, ofthe TSG101 protein, with the agent and measuring MDM2 level in the cell;and (c) comparing MDM2 levels measured in (a) and (b). A difference inMDM2 levels as determined in step (c) identifies the agent as capable ofmodulating the interaction of the TSG101 protein with MDM2.

The invention also provides methods of modulating a level of MDM2 or p53in a cell. The methods comprise contacting the cell with a polypeptideor derivative thereof that comprises a polypeptide comprising anubiquitination-regulating domain.

The invention also provides methods of modulating a level of MDM2, orTSG101, or p53 in a cell. The methods comprise contacting the cell withan agent that is capable of modulating the interaction of a TSG101protein with MDM2.

The invention further provides methods for treating a subject of adisease or any other undesirable conditions that are a result of adeviation of a level of TSG101, or MDM2, or p53 from normal ranges. Thesubject that can be treated includes a human or a non-human mammal.

In one embodiment, the invention provides methods for treating a subjectof a condition resulting from a change in a level of MDM2 protein incells of the subject. The methods comprise administering to the subjecta therapeutically effective amount of an agent which comprises anubiquitination-regulating domain.

In another embodiment, the invention provides methods of treating asubject of a condition resulting from a change in a level of a TSG101protein in cells of the subject. The methods comprising administering tothe subject a therapeutically effective amount of an agent, said agentmodulating the interaction of said TSG101 protein with MDM2.

In still another embodiment, the invention provides methods fortreatment of a proliferative disease in a subject comprising: (a)monitoring the subject for a level of p53; and (b) treating the subjectwith an agent which comprises an ubiquitination-regulating domain so asto maintain the level of p53 within a target range.

The invention also provides methods for treating a subject of aproliferative disease. The methods comprise administering to the subjecta therapeutically effective amount of an agent that is capable ofmodulating the interaction between TSG101 and MDM2. In a specificembodiment, the invention provides a method for treatment of aproliferative disease in a subject comprising: (a) monitoring thesubject for a level of TSG101; and (b) treating the subject with anagent which is capable of modulating the interaction of the TSG101 withMDM2 so as to maintain the level of TSG101 within a target range.

In any of the methods of the invention where anubiquitination-regulating domain is used, the ubiquitination-regulatingdomain can be an ubiquitination-regulating domain, or a functionalfragment thereof, of a TSG101 protein, e.g., a human TSG101 protein. Ina preferred embodiment, the ubiquitination-regulating domain comprisesamino acid residues 1-140 of a human TSG101 protein, e.g., amino acidresidues 1-140 of SEQ ID NO:1. In another preferred embodiment, theubiquitination-regulating domain comprises amino acid residues 50-140 ofa human TSG101, e.g., amino acid residues 50-140 of SEQ ID NO:1. Inanother preferred embodiment, the ubiquitination-regulating domaincomprises amino acid residues 140-250 of a human TSG101 protein, e.g.,amino acid residues 140-250 of SEQ ID NO:1. In still other embodiments,the ubiquitination-regulating domain may comprises amino acid residues10-140, 20-140, 30-140, 40-140, 1-160, 1-180, 1-200, 1-220, 50-250 or1-250 of a human TSG101, e.g., amino acid residues 10-140, 20-140,30-140, 40-140, 1-160, 1-180, 1-200, 1-220, 50-250 or 1-250 of SEQ IDNO:1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D depict the results of Western blot analysis of theinteraction of TSG101 with p53 and MDM2.

FIGS. 2A-E depict the results of experiments showing the effect ofexpression of TSG101 on the cellular level of p53 and the degradation ofMDM2.

FIGS. 3A and 3B depict the results of experiments showing the effects ofUbc domains of TSG101 on MDM2 degradation and ubiquitination.

FIGS. 4A-4F depict the results of experiments showing MDM2-dependentproteolysis of TSG101.

FIG. 5 depicts a model showing functional interactions of theTSG101/MDM2 and p53/MDM2 feedback control loops.

FIG. 6 depicts the 390 amino acid sequence of human TSG101 protein (SEQID NO:1). (GenBank® Accession No. U82130.1/GI: 1772663)

DETAILED DESCRIPTION OF THE INVENTION

Before the present invention is described, it is to be understood thatthis invention is not limited to particular embodiments described, assuch may, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present invention will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference to disclose and describe the methodsand/or materials in connection with which the publications are cited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “acell” includes a plurality of such cells and reference to “thepolynucleotide” includes reference to one or more polynucleotides andequivalents thereof known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DEFINITIONS

The terms “polynucleotide” and “nucleic acid molecule” are usedinterchangeably herein to refer to polymeric forms of nucleotides of anylength. The polynucleotides may contain deoxyribonucleotides,ribonucleotides, and/or their analogs. Nucleotides may have anythree-dimensional structure, and may perform any function, known orunknown. The term “polynucleotide” includes single-, double-stranded andtriple helical molecules. “Oligonucleotide” generally refers topolynucleotides of between about 5 and about 100 nucleotides of single-or double-stranded DNA. However, for the purposes of this disclosure,there is no upper limit to the length of an oligonucleotide.Oligonucleotides are also known as oligomers or oligos and may beisolated from genes, or chemically synthesized by methods known in theart.

The following are non-limiting embodiments of polynucleotides: a gene orgene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA,recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes, and primers. A nucleic acid molecule may alsocomprise modified nucleic acid molecules, such as methylated nucleicacid molecules and nucleic acid molecule analogs. Analogs of purines andpyrimidines are known in the art. Nucleic acids may be naturallyoccurring, e.g. DNA or RNA, or may be synthetic analogs, as known in theart. Such analogs may be preferred for use as probes because of superiorstability under assay conditions. Modifications in the native structure,including alterations in the backbone, sugars or heterocyclic bases,have been shown to increase intracellular stability and bindingaffinity. Among useful changes in the backbone chemistry arephosphorothioates; phosphorodithioates, where both of the non-bridgingoxygens are substituted with sulfur; phosphoroamidites; alkylphosphotriesters and boranophosphates. Achiral phosphate derivativesinclude 3′-O′-5′-S-phosphorothioate, 3′-S-5′-O-phosphorothioate,3′-CH2-5′-O-phosphonate and 3′-NH-5′-O-phosphoroamidate. Peptide nucleicacids replace the entire ribose phosphodiester backbone with a peptidelinkage.

Sugar modifications are also used to enhance stability and affinity. Theα-anomer of deoxyribose may be used, where the base is inverted withrespect to the natural β-anomer. The 2′-OH of the ribose sugar may bealtered to form 2′-O-methyl or 2′-O-allyl sugars, which providesresistance to degradation without comprising affinity.

Modification of the heterocyclic bases must maintain proper basepairing. Some useful substitutions include deoxyuridine fordeoxythymidine; 5-methyl-2′-deoxycytidine and 5-bromo-2′-deoxycytidinefor deoxycytidine. 5-propynyl-2′-deoxyuridine and5-propynyl-2′-deoxycytidine have been shown to increase affinity andbiological activity when substituted for deoxythymidine anddeoxycytidine, respectively.

The terms “polypeptide” and “protein”, used interchangeably herein,refer to a polymeric form of amino acids of any length, which caninclude coded and non-coded amino acids, chemically or biochemicallymodified or derivatized amino acids, and polypeptides having modifiedpeptide backbones. The term includes fusion proteins, including, but notlimited to, fusion proteins with a heterologous amino acid sequence,fusions with heterologous and homologous leader sequences, with orwithout N-terminal methionine residues; immunologically tagged proteins;and the like.

As used herein the term “isolated” is meant to describe a compound ofinterest that is in an environment different from that in which thecompound naturally occurs. “Isolated” is meant to include compounds thatare within samples that are substantially enriched for the compound ofinterest and/or in which the compound of interest is partially orsubstantially purified.

As used herein, the term “substantially purified” refers to a compoundthat is removed from its natural environment and is at least 60% free,preferably 75% free, and most preferably 90% free from other componentswith which it is naturally associated.

The term “treatment” is used herein to encompass any treatment of anydisease or condition in a mammal, particularly a human, and includes: a)preventing a disease, condition, or symptom of a disease or conditionfrom occurring in a subject which may be predisposed to the disease buthas not yet been diagnosed as having it; b) inhibiting a disease,condition, or symptom of a disease or condition, e.g., arresting itsdevelopment and/or delaying its onset or manifestation in the patient;and/or c) relieving a disease, condition, or symptom of a disease orcondition, e.g., causing regression of the condition or disease and/orits symptoms.

By “subject” or “individual” or “patient” is meant any mammalian subjectfor whom diagnosis or therapy is desired, particularly humans. Othersubjects may include cattle, dogs, cats, guinea pigs, rabbits, rats,mice, horses, and so on.

For simplicity reasons, this disclosure often makes references to a geneor protein by its generic name. In such cases, it will be understoodthat the disclosure is equally applicable to any mammalian homologs ofthe gene or protein. This disclosure also often makes references to agene or protein by its name for a specific species. In such cases,unless specifically indicated, it will be understood that the disclosureis equally applicable to any other mammalian homologs of the gene orprotein. For example, unless specifically indicated, MDM2 will beunderstood to encompass any mammalian homolog of the MDM2 protein.

Overview

The p53 tumor suppressor protein and the MDM2 oncoprotein form afeedback-control loop that up-regulates cellular MDM2 production, blocksp53 activity, and promotes p53 decay. Tsg101 was discovered as a genewhose deficiency results in neoplastic transformation of NIH3T3 cellsand the ability to generate metastatic tumors in nude mice. Its proteinproduct contains a domain, Ubc, characteristic of the catalytic domainof ubiquitin conjugase (E2) enzymes but lacking an active site cysteincrucial for ubiquitin conjugase activity. Defective regulation ofubiquitination has been implicated in diseases includingneurodegenerative diseases, autoimmune diseases, developmentalabnormalities, metabolic disease and cancers (see, e.g., reference 41).The amino residues 140-250 of TSG101 includes a proline-rich domainwhich has been suggested to be a binding site for other proteins, suchas NEDD4 (a developmentally regulated ubiquitin-protein ligase). NEDD4targets the epithelial sodium channel (ENaC), a key regulator of bloodsodium concentration, for ubiquitin-mediated turnover (see, Staub et.al., EMBO J. 1996, 15:2371-80). Here we report that anubiquitination-regulating domain, e.g., the Ub domain of a TSG101,regulates ubiquitination in a cell. Such regulation may play a role in,e.g., regulation of ubiquitin-mediated proteolysis, translation, DNArepair, activation of transcription of factors and kinases, andtranslocation (see, e.g., references 40, 41). For example, we show thatTSG101 participates with MDM2 in a separate autoregulatory loop thatmodulates the cellular levels of both proteins, and also of p53, byaffecting protein decay. We show that the Ub domain, or a functionalfragment thereof (e.g., a biologically-active fragment), of TSG101interferes with ubiquitination of MDM2, that TSG101 inhibits MDM2 decayand elevates its steady-state level, and that these events areassociated with down regulation of p53 protein. Conversely, pulse chaseand Western blot experiments in wild type and mutant fibroblastsindicate that elevation of MDM2 by overexpression of wild type p53, byamplification of the endogenous MDM2 gene, or by transfection ofMDM2-expressing constructs promotes TSG101 loss, which we show occurs by26S proteasome-dependent decay. Our results identify TSG101 as both aregulator of, and target of, MDM2/p53 circuitry.

Specific aspects of the invention will now be described in more detail.

Polypeptide Compositions

In some embodiments, the invention provides isolated polypeptidescomprising a ubiquitination-regulating domain. As used herein, anubiquitination-regulating domain refers to a polypeptide which regulatesubiquitination, e.g., via regulating ubiquitin conjugases (E2 enzymes).In some of these embodiments, the ubiquitination-regulating domain hasthe amino acid sequence of an ubiquitination-regulating domain of aTSG101, e.g., an ubiquitination-conjugase-like Ubc domain of a humanTSG101. In some embodiments, the ubiquitination-regulating domain hasthe amino acid sequence of a functional fragment of anubiquitination-regulating domain of a TSG101, e.g., a human TSG101 (SEQID NO:1; GenBank Accession No. U82130.1/GI: 1772663). As used herein, afunctional fragment of an ubiquitination-conjugase-like Ubc domainrefers to any fragment of the Ub domain that regulates ubiquitination.Fragments having such activity are readily determined by, e.g., methodsas described in the application. In one embodiment, theubiquitination-regulating domain comprises amino acid residues 1-140 ofa human TSG101, e.g., amino acid residues 1-140 of SEQ ID NO:1. Inanother embodiment, the ubiquitination-regulating domain comprises aminoacid residues 50-140 of a human TSG101, e.g., amino acid residues 50-140of SEQ ID NO:1. In still another embodiment, theubiquitination-regulating domain comprises amino acid residues 140-250of a human TSG101, e.g., amino acid residues 140-250 of SEQ ID NO:1. Instill other embodiments, the ubiquitination-regulating domain maycomprises, e.g., amino acid residues 10-140, 20-140, 30-140, 40-140,1-160, 1-180, 1-200, 1-220, 50-250 or 1-250 of a human TSG101, e.g.,amino acid residues 10-140, 20-140, 30-140, 40-140, 1-160, 1-180, 1-200,1-220, 50-250 or 1-250 of SEQ ID NO:1. The nucleotide and amino acidsequences of TSG101 are also disclosed in U.S. Pat. No. 5,891,668. Theconstruct designated TSG101B in the Examples comprises an insert that islargely a TSG101 Ubc domain, and is an example of anubiquitination-regulating domain. Alternatively, anubiquitination-regulating domain may be a Ubc domain from an ubiquitinconjugase that is altered to lack the active site cysteine that iscritical for the function of a ubiquitin conjugase.

Production of Subject Polypeptides

The subject polypeptides can be produced synthetically, or can beproduced recombinantly, i.e., a polynucleotide comprising a codingregion encoding a polypeptide comprising an ubiquitination-regulatingdomain can be inserted into an expression vector, and theubiquitination-regulating domain coding region transcribed andtranslated.

Ubiquitination-regulating domain-containing polypeptides can be producedsynthetically, using any known method. One may employ solid phasepeptide synthesis techniques, where such techniques are known to thoseof skill in the art. See Jones, The Chemical Synthesis of Peptides(Clarendon Press, Oxford)(1994). Generally, in such methods a peptide isproduced through the sequential addition of activated monomeric units toa solid phase bound growing peptide chain.

For expression, an expression cassette may be employed. The expressionvector will provide a transcriptional and translational initiationregion, which may be inducible or constitutive, where the coding regionis operably linked under the transcriptional control of thetranscriptional initiation region, and a transcriptional andtranslational termination region. These control regions may be native tothe subject gene, or may be derived from exogenous sources.

Expression vectors generally have convenient restriction sites locatednear the promoter sequence to provide for the insertion of nucleic acidsequences encoding heterologous proteins. A selectable marker operativein the expression host may be present. Expression vectors may be usedfor the production of fusion proteins, where the exogenous fusionpeptide provides additional functionality, i.e. increased proteinsynthesis, stability, reactivity with defined antisera, an enzymemarker, e.g. β-galactosidase, etc.

Expression cassettes may be prepared comprising a transcriptioninitiation region, the polynucleotide comprising a nucleotide sequenceencoding a polypeptide comprising a ubiquitination-regulating domain,and a transcriptional termination region. After introduction of the DNA,the cells containing the construct may be selected by means of aselectable marker, the cells expanded and then used for expression.

The polypeptides may be expressed in prokaryotes or eukaryotes inaccordance with conventional ways, depending upon the purpose forexpression. For large scale production of the protein, a unicellularorganism, such as E. coli, B. subtilis, S. cerevisiae, insect cells incombination with baculovirus vectors, or cells of a higher organism suchas vertebrates, particularly mammals, e.g. COS 7 cells, may be used asthe expression host cells. In some situations, it is desirable toexpress the gene in eukaryotic cells, where the protein will benefitfrom native folding and post-translational modifications. Small peptidescan also be synthesized in the laboratory. Polypeptides that are subsetsof the complete amino acid sequence may be used to identify andinvestigate parts of the protein important for function, or to raiseantibodies directed against these regions.

With the availability of the protein or fragments thereof in largeamounts, by employing an expression host, the protein may be isolatedand purified in accordance with conventional ways. A lysate may beprepared of the expression host and the lysate purified using HPLC,exclusion chromatography, gel electrophoresis, affinity chromatography,or other purification technique.

The invention further provides polynucleotides comprising a nucleotidesequence encoding a polypeptide comprising a ubiquitination-regulatingdomain, as well as recombinant vectors (“constructs”) comprising suchpolynucleotides. Recombinant vectors are useful for propagation of thesubject polynucleotides (cloning vectors). They are also useful foreffecting expression of a subject polynucleotide in a cell (expressionvectors). Some vectors accomplish both cloning and expression functions.The choice of appropriate vector is well within the skill of the art.Many such vectors are available commercially.

The invention further provides a host cell comprising a subjectrecombinant vector. The present invention further provides host cells,which may be isolated host cells, comprising a polynucleotide of theinvention. Suitable host cells include prokaryotes such as E. coli, B.subtilis, eukaryotes, including insect cells in combination withbaculovirus vectors, yeast cells, such as Saccharomyces cerevisiae, orcells of a higher organism such as vertebrates, including amphibians(e.g., Xenopus laevis oocytes), and mammals, particularly mammals, e.g.COS cells, CHO cells, 293 cells, 3T3 cells, and the like, may be used asthe expression host cells. Host cells can be used for the purposes ofpropagating a subject polynucleotide, for production of a subjectpolypeptide, or in cell-based methods for identifying agents whichmodulate a level of subject mRNA and/or protein and/or activity in acell.

Antibodies to an Ubiquitination-Regulating Domain

An ubiquitination-regulating domain of the invention may be used as animmunogen to generate antibodies which immunospecifically bind such animmunogen. Such antibodies include but are not limited to polyclonal,monoclonal, chimeric, single chain, Fab fragments, and an Fab expressionlibrary. Such antibodies are useful in modulating the interactionbetween the ubiquitination-regulating domain and other proteins in acell, e.g., an MDM2 protein and/or a p53 protein. In a specificembodiment, antibodies to an ubiquitination-regulating domain comprisingthe ubiquitination-regulating domain, or a functional fragment thereof,of a human TSG101 protein are produced. In another embodiment,antibodies to a polypeptide comprising amino residues 1-140 of a humanTSG101 protein, e.g., amino acid residues 1-140 of SEQ ID NO:1, areproduced. In yet another embodiment, antibodies to a polypeptidecomprising amino residues 140-250 of a human TSG101 protein, e.g., aminoacid residues 140-250 of SEQ ID NO:1, are produced. In still anotherembodiment, antibodies to a polypeptide comprising amino residues 50-140of a human TSG101 protein, e.g., amino acid residues 50-140 of SEQ IDNO:1, are produced.

Various procedures known in the art may be used for the production ofpolyclonal antibodies to an ubiquitination-regulating domain. In aparticular embodiment, rabbit polyclonal antibodies to anubiquitination-regulating domain, e.g., the ubiquitination-regulatingdomain, or a subsequence thereof, of a human TSG101 protein can beobtained. For the production of antibody, various host animals can beimmunized by injection with a native ubiquitination-regulating domain,or a synthetic version, or derivative (e.g., fragment) thereof,including but not limited to rabbits, mice, rats, etc. Various adjuvantsmay be used to increase the immunological response, depending on thehost species, and including but not limited to Freund's (complete andincomplete), mineral gels such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanins, dinitrophenol, andpotentially useful human adjuvants such as BCG (bacille Calmette-Guerin)and corynebacterium parvum.

For preparation of monoclonal antibodies directed to anubiquitination-regulating domain, or a fragment thereof, any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture may be used. For example, the hybridoma techniqueoriginally developed by Kohler and Milstein (1975, Nature 256:495-497),as well as the trioma technique, the human B-cell hybridoma technique(Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridomatechnique to produce human monoclonal antibodies (Cole et al., 1985, inMonoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.77-96). In an additional embodiment of the invention, monoclonalantibodies can be produced in germ-free animals utilizing recenttechnology (see e.g., PCT/US90/02545). According to the invention, humanantibodies may be used and can be obtained by using human hybridomas(Cole et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030) or bytransforming human B cells with EBV virus in vitro (Cole et al., 1985,in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, pp. 77-96).In fact, according to the invention, techniques developed for theproduction of “chimeric antibodies” (Morrison et al., 1984, Proc. Natl.Acad. Sci. U.S.A. 81:6851-6855; Neuberger et al., 1984, Nature312:604-608; Takeda et al., 1985, Nature 314:452-454) by splicing thegenes from a mouse antibody molecule specific for a humanubiquitination-regulating domain together with genes from a humanantibody molecule of appropriate biological activity can be used; suchantibodies are within the scope of this invention.

According to the invention, techniques described for the production ofsingle chain antibodies (U.S. Pat. No. 4,946,778) can be adapted toproduce ubiquitination-regulating domain-specific single chainantibodies. An additional embodiment of the invention utilizes thetechniques described for the construction of Fab expression libraries(Huse et al., 1989, Science 246:1275-1281) to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificityfor an ubiquitination-regulating domain.

Antibody fragments which contain the idiotype of the molecule can begenerated by known techniques. For example, such fragments include butare not limited to: the F(ab′)₂ fragment which can be produced by pepsindigestion of the antibody molecule; the Fab′ fragments which can begenerated by reducing the disulfide bridges of the F(ab′)₂ fragment, theFab fragments which can be generated by treating the antibody moleculewith papain and a reducing agent, and Fv fragments.

In the production of antibodies, screening for the desired antibody canbe accomplished by techniques known in the art (e.g., enzyme-linkedimmunosorbent assay or ELISA). For example, to select antibodies whichrecognize a specific domain of an ubiquitination-regulating domain, onemay assay generated hybridomas for a product which binds to anubiquitination-regulating domain containing such domain.

Antibodies specific to an epitope of an ubiquitination-regulating domainare also provided.

The foregoing antibodies can be used in methods known in the artrelating to the activity of an ubiquitination-regulating domain of theinvention, e.g., for determination interaction partners of theseproteins, measuring levels thereof in appropriate physiological samples,in diagnostic methods, etc.

Compositions

The present invention further provides compositions, includingpharmaceutical compositions, comprising the polypeptides,polynucleotides, agents, recombinant vectors, and host cells of theinvention. These compositions may include a buffer, which is selectedaccording to the desired use of the polypeptide, agent, polynucleotide,recombinant vector, or host cell, and may also include other substancesappropriate to the intended use. Those skilled in the art can readilyselect an appropriate buffer, a wide variety of which are known in theart, suitable for an intended use. In some instances, the compositioncan comprise a pharmaceutically acceptable excipient, a variety of whichare known in the art and need not be discussed in detail herein.Pharmaceutically acceptable excipients have been amply described in avariety of publications, including, for example, A. Gennaro (1995)“Remington: The Science and Practice of Pharmacy”, 19th edition,Lippincott, Williams, & Wilkins.

Methods

The invention provides methods for modulating mammalian cellproliferation; methods of inhibiting interaction of TSG101 with MDM2 ina mammalian cell; methods of modulating the rate of decay of MDM2 in acell; methods of modulating a level of p53 polypeptide in a cell;methods of inhibiting ubiquitination of a polypeptide in a cell; andmethods of maintaining a level of TSG101 in a cell.

Screening Assays

The present invention provides screening methods for identifying agents,e.g., antibodies, which modulate a TSG101 interaction with MDM2 in acell.

As used herein, the term “modulate” encompasses “increase” and“decrease”. In some embodiments, agents which reduce TSG 101/MDM2interaction in a cell are of interest. Such agents may be of interest ascandidates for promoting cell division, reducing apoptosis, or reducinggrowth arrest.

The terms “agent”, “substance” and “compound” are used interchangeablyherein. Candidate agents encompass numerous chemical classes, which maybe synthetic, semi-synthetic, or naturally-occurring inorganic ororganic molecules. Candidate agents may be small organic compoundshaving a molecular weight of more than 50 and less than about 2,500daltons. Candidate agents may comprise functional groups necessary forstructural interaction with proteins, particularly hydrogen bonding, andtypically include at least an amine, carbonyl, hydroxyl or carboxylgroup, and may contain at least two of the functional chemical groups.The candidate agents may comprise cyclical carbon or heterocyclicstructures and/or aromatic or polyaromatic structures substituted withone or more of the above functional groups. Candidate agents are alsofound among biomolecules including peptides, saccharides, fatty acids,steroids, purines, pyrimidines, derivatives, structural analogs orcombinations thereof.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides and oligopeptides. Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant and animal extractsare available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs.

Where the screening assay is a binding assay, one or more of themolecules may be joined to a label, where the label can directly orindirectly provide a detectable signal. Various labels includeradioisotopes, fluorescers, chemiluminescers, enzymes, specific bindingmolecules, particles, e.g. magnetic particles, and the like. Specificbinding molecules include pairs, such as biotin and streptavidin,digoxin and antidigoxin etc. For the specific binding members, thecomplementary member would normally be labeled with a molecule thatprovides for detection, in accordance with known procedures.

A variety of other reagents may be included in the screening assay.These include reagents like salts, neutral proteins, e.g. albumin,detergents, etc that are used to facilitate optimal protein-proteinbinding and/or reduce non-specific or background interactions. Reagentsthat improve the efficiency of the assay, such as protease inhibitors,nuclease inhibitors, anti-microbial agents, etc. may be used. Themixture of components are added in any order that provides for therequisite binding. Incubations are performed at any suitabletemperature, typically between 4° C. and 40° C. Incubation periods areselected for optimum activity, but may also be optimized to facilitaterapid high-throughput screening. Typically between 0.1 and 1 hour willbe sufficient.

The present invention also provides methods for identifying cellularproteins that interact with an ubiquitination-regulating domain. Anymethods known in the art, including but are not limited to, yeasttwo-hybrid assays (see, e.g., Fields and Song, 1989, Nature 340:245-246and U.S. Pat. No. 5,283,173), immunoprecipitation, Western blot (see,example section below), can be used for this purpose.

Such screening methods may be performed, e.g., on cell lines or inorganisms expressing various levels of TSG101. In this regard, TSG101heterozygous and homozygous knockout mice as described (38) may be used.

Examples

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric.

Materials and Methods

Plasmid and Vector Construction. Full-length human TSG101 cDNA wasinserted into the pLLEXP1 vector (1) between the cytomegaloviruspromoter and polyadenylation site. HA-tagged (human influenzahemagglutinin peptide, YPYDVPDY), Flag-tagged and c-Myc tagged TSG101and TSG101 deletion mutant cDNAs were generated by PCR and were alsocloned using pLLEXP1. Vectors expressing human wild type p53 (pC53-SN3)(25), human mutant p53 (pC53-Cx21an 3, aa 175 mutation, Arg to His)(26), human MDM2 (pCHDM1B) (27), and HM-Ub and HM-K48R-Ub (28) have beendescribed. pCMV-GFP (Clontech) was used to express green fluorescentprotein (GFP).

Cell Culture and Transfection. Saos-2, U2OS, SJSA-1 and NIH3T3 cellswere obtained from the American Type Culture Collection (ATCC). Cellswere cultured in DMEM (Saos-2, NIH3T3, p53−/− MEF, and p53−/− andMDM2−/− MEF; ref. (29) or RPMI (U2OS and SJSA-1) supplemented with 10%fetal bovine serum. Transfections were carried out using eitherLipofectamine (Life Technologies) or FuGENE™6 (Roche) as described bythe manufacturer.

Immunoprecipitation and Western blot analysis. Immunoprecipitation andWestern blot analysis were performed as described (30). Cells were lysedwith NP-40 lysis buffer on ice, and the protein extracts were preclearedwith prewashed Pierce Protein A/G agarose beads (50 μl of 50% slurry per0.5 ml protein extract). The pre-cleared protein extracts were incubatedwith antibody for 8 hours to overnight at 4 C° on a rotating rocker, andthen with the prewashed Pierce Protein A/G agarose beads for anadditional 2 hours (10-15 μl of 50% slurry per 0.5 ml protein extract).The immunocomplex was washed 4 times with NP-40 buffer, dissolved in SDSloading buffer and fractionated on 10% SDS-polyacrylamide gels(BIO-RAD). The proteins were then transferred to NitroPure membrane(MSI) and incubated with specific antibody and appropriatehorseradish-peroxidase-coupled secondary antibody (Santa Cruz andPromega). The membranes were washed in phosphate buffered saline (PBS)and visualized with ECL (Santa Cruz). Autoradiograms of Western blotswere scanned with Scanmaster 3tm (Howtek) and analyzed using theQuantity One program (pdi). Antibodies used for immunoprecipations wererabbit anti-TSG101 (1:200, Clontech), anti-p53 (Ab-1, 1:400,Calbiochem), anti-MDM2 (SMP-14, 1:200, Santa Cruz Biotechnology), andanti-hemagglutinin (HA) (1:200, Clontech). Antibodies used for Westernblots were rabbit anti-TSG101 (1:200, Clontech), anti-p53 (DO-1, 1:1000,Santa Cruz Biotechnology), anti-hemagglutinin (HA) (1:500, horseradishperoxidase (HRP)-labeled, Roche) and anti-Flag (1:500, M2, Kodak),anti-α-tubulin (1:20000, Neomark), anti-rabbit IgG (1:5000, HRP-labeled,Promega) and anti-mouse IgG (1:10,000, HRP-labeled, Santa CruzBiotechnology). Anti-GFP antibody was obtained from Clontech and wasused at 1:500 dilution.in vivo Ubiquitination of MDM2. HM-Ub or HM-K48R-Ub was introduced intoSJSA-1 cells by cotransfection with vectors expressing TSG101 mutantproteins B or F or with controls lacking an insert or expressing TSG101cDNA in the antisense direction. Twenty-four hours later, thetransfected cell cultures were treated with MG132 (2 μM) for 12additional hours, lysed with 6 M guanidinium HCl, and sonicated for 20s. The His-tagged proteins were purified using Ni-NTA spin columns(Qiagen), washed 4 times with 0.8 ml wash buffer (8 M Urea, 0.1 MNaH2PO4, 0.01 M Tris Cl, pH 6.2), and eluted once with wash buffer at pH4, and an additional time with pH4 wash buffer containing 250 mMimidazole. The purified proteins were analyzed by Western blotting asabove.Pulse-Chase Experiments. NIH 3T3, p53^(−/−) MEF, andp53^(−/−)/MDM2^(−/−) MEF cells (1×10⁶) were seeded onto 100 mm platesfor 24 hours and were pulse-labeled with [³⁵S]methionine for 2 hours,washed twice with prewarmed PBS, and chased by culturing in DMEMsupplemented with 10% fetal bovine serum for 0, 2, 4, 8, 12 hours. Celllysates from pulse-labeled cells were immunoprecipitated withanti-TSG101 antibody (10 μg/500 μg total protein), resolved byelectrophoresis in 10% SDS-polyacrylamide gels, and analyzed using aphosphoimager (ImageQuant Storm 840, Molecular Dynamics).

Results

Physical and functional interaction of TSG101 with p53 and MDM2. Duringinvestigations aimed at identifying physical and functional interactionsbetween TSG101 and proteins previously implicated in tumorigenesis, wefound that TSG101 can bind to both p53 and MDM2. This is shown in FIG.1, which presents results of Western blot analyses of extracts of Saos-2human osteosarcoma cells co-transfected with constructs expressingcombinations of these proteins. Constructs expressing human TSG101, p53,or MDM2 proteins (2 μg of DNA for each plasmid) were introduced bytransfection into Saos-2 cells, as indicated in Methods. Proteinextracts from transfected cell populations were immunoprecipitated bythe antibodies indicated. IP: immunoprecipitation, IB: immunoblotting.(a) Native or HA-tagged TSG101 or p53 proteins in Western blots weredetected by anti-p53 monoclonal antibody (AB-1; 1:1000; the secondaryantibody was goat anti-mouse HRP, diluted 1:1000) or (b) by anti-HAantibody labeled with HRP (diluted 1:500). (c) and (d) Western blotdetection of immunoprecipitated proteins analyzed with anti-MDM2antibody.

Complexes immunoprecipitated from cell extracts by antibody to TSG101contained p53, either untagged or as fused to an influenza Bhemagglutinin [HA] peptide epitope used for detection, and conversely,identified TSG101 in complexes immunoprecipitated with antibody to p53(FIGS. 1 a and b). Similarly, a 90 kD band detected with anti-MDM2antibody and representing an MDM2 complex with the small ubiquitin-likeprotein, SUMO-1 (24) was present in Saos-2 cell protein complexesimmunoprecipitated with antibodies to native or epitope-tagged TSG101(FIGS. 1 c and d).

MDM2 is a ubiquitin protein ligase that mediates its own decay as wellas the degradation of p53 (31, 32). That the ability of TSG101 tointeract with MDM2 and/or p53 has functional consequences on the steadystate levels of MDM2 and p53 is shown in FIG. 2: FIG. 2( a): Theindicated constructs overexpressing p53 and HA-TSG101 were introducedinto Saos-2 cells by transfection and p53 and HA-TSG101 levels wereanalyzed by Western blotting 48 hours later. + indicates 25 ng oftransfected p53 expression vector DNA or 4 μg of HA-TSG101 expressionvector DNA, ++ indicates 8 μg of HA-TSG101 expression vector DNA. Thedensity of p53 bands relative to cellular α-tubulin was determined byscanning of exposed films. FIG. 2( b): The identical constructsoverexpressing p53 and HA-TSG101 were introduced intop53^(−/−)/MDM2^(−/−) MEF cells by transfection and p53 and HA-TSG101levels were analyzed as in a.

TSG101 overexpression has divergent effects on the steady-state cellularlevels of both the p53 and MDM2 proteins. As seen in FIG. 2 a,overproduction of TSG101 in Saos-2 cells, which synthesize endogenousMDM2 protein but not native p53, reduced the level of p53 expressed froma transfected construct by 70%. However, in identically transfectedcells that carry null chromosomal mutations in both p53 and MDM2 andthus lack the ability to synthesize either of these proteins [i.e.,p53^(−/−)/MDM2^(−/−) mouse embryo fibroblasts (MEF) (29, 33, 34); FIG. 2b], we observed no effect of TSG101 on p53—suggesting thatTSG101-mediated reduction of the p53 level requires the presence ofMDM2.

Although less than 50% of the population of Saos-2 cells was transfectedby TSG101-expressing constructs under the experimental conditions weemployed (data not shown), this was sufficient to elevate the MDM2 levelin extracts of the entire cell population (FIG. 2 a), supporting thenotion that TSG101 down regulates p53 by elevating MDM2. Direct evidencefor this conclusion was provided by experiments in which Saos-2 cellswere transfected with constructs expressing p53, MDM2, and TSG101,individually or in combination. FIG. 2( c): Constructs overexpressingthe proteins indicated were introduced into Saos-2 cells by transfectionand p53 and MDM2 levels were analyzed as in a. + indicates 25 ng oftransfected p53 expression vector DNA, 1 μg of MDM2 expression vectorDNA, 4 μg of TSG101 expression vector DNA. Overproduction of MDM2 from aco-transfected CMV-based expression vector resulted in a decrease in thelevel of p53 protein (FIG. 2 c, lanes 1-2); concurrent overproduction ofTSG101 in co-transfected cells was associated with further elevation ofthe MDM2 protein level (FIG. 2 c, lanes 3 and 5) and a prominent furtherdecrease in p53 (lane 3).

Effect of TSG101 on MDM2 decay. MDM2 normally has a short half-life of15-20 minutes (35). FIG. 2( d): Saos-2 cells were transfected by anMDM2-expressing construct in the absence or presence of aTSG101-expressing construct. Cellular protein was extracted afteraddition of cyclohexamide at the indicated times and analyzed by Westernblotting as in (a) using anti-MDM2 antibody. A GFP expression vector wasco-transfected to normalize transfection efficiency; the expressed GFPprotein was detected with anti-GFP antibody. FIG. 2( e): Plot ofdegradation of MDM2 for the experiment shown in (d), which wasrepresentative of five separate experiments. As seen in FIGS. 2 d and e,TSG101 inhibits MDM2 degradation and prolongs its half-life. In thisexperiment, cells transfected with an construct expressing MDM2 from aCMV promoter, or co-transfected with constructs that express both MDM2and TSG101, were treated with cyclohexamide to stop protein synthesis,and MDM2 protein was assayed by Western blot analysis of samples takenat the indicated times. The half-life observed for MDM2 (approximately15 min), which is consistent with earlier determinations (35), nearlydoubled (to 28 min) in cells that concurrently overexpressed TSG101.The ubiquitin-conjugase-like Ubc domain of TSG101 inhibitsubiquitination of MDM2. Because the Ub domain of TSG101 lacks a cysteineresidue required for conjugase function (12-14), it previously wasspeculated that TSG101 may inhibit ubiquitination by formingnon-productive complexes with ubiquitin or its target proteins andconsequently interfering with the function of bona fide E2 (12, 13). Theresults seen in FIG. 3 a, which show the effects of mutant TSG101proteins on the steady state level of MDM2 expressed from aco-transfected construct, indicate that TSG101's ability to stabilizeMDM2 is sharply reduced by deletion of sequences from the Ub domain.They also show that overexpression of the TSG101 Ubc domain's ‘a’region, which contains residues bracketing the ‘active site’ locus thatin functional E2 enzymes contains a cystein, is sufficient to causeaccumulation of MDM2 (construct A). The presence of the ‘b’ region ofTSG101's Ubc domain enhanced the effects of the ‘a’ region and even inthe absence of Ubc_(a) led to some stabilization of MDM2 (constructs Bvs. A and E vs. F).

Further analysis of truncated TSG101 proteins consisting of largely theUb domain (construct TSB101B) or lacking this domain (construct TSG101F)showed that overexpression of the TSG101 Ubc domain interferes withubiquitination of endogenous MDM2 (FIG. 3 b). In the experiment shown inthe left panel, cellular proteins conjugated to His-tagged ubiquitinwere isolated by Ni-NTA column chromatography, and MDM2 was identifiedin this protein pool by Western blotting using anti-MDM2 antibody.Notwithstanding the ability of TSG101 and its Ubc domain to globallyincrease the cellular level of both endogenous and adventitious MDM2(FIG. 2), cells overproducing the TSG101 Ubc domain showed a decrease inubiquitinated MDM2 vs. controls (FIG. 3 b left panel, lanes 1 and 2).That the TSG101 Ubc domain can decrease ubiquitination of MDM2 wasdemonstrated also by an experiment in which ubiquitin chains initiatedon cellular proteins were tagged with a Ub variant (K48R) (28) thatimpedes ubiquitin chain elongation; this preserves tagged proteins andallows the extent of Ub addition to endogenous MDM2 to be evaluated byimmunoprecipitation of MDM2 (FIG. 3 b, right panel).

FIG. 3( a) The indicated constructs expressing c-Myc tagged full-lengthand deletion mutants of TSG101 (A-F, 8 μg), a construct expressingHA-tagged MDM2 (1.5 μg), and a construct expressing GFP (2 ng) wereintroduced into U2OS cells by transfection. HA-MDM2, c-Myc taggedTSG101s (A-F) and GFP were detected by Western blotting 48 hours aftertransfection with anti-HA, anti-c-Myc and anti-GFP antibodies. (b) Theindicated constructs express ubiquitin tagged with both His₆ and c-Myc(HM-Ub, 5 μg, left panel), or a dominant negative variant ubiquitintagged with His₆ and c-Myc (HM-K48R-Ub, 5 μg, right panel). These wereco-transfected into SJSA-1 cells with a GFP expression and construct (2ng), constructs expressing TSG101 mutant B or F (4 μg each), a constructexpressing antisense TSG101(4 μg), or a construct containing no DNAinsert (4 μg). Protein extracts were applied to Ni-NTA columns and theubiquitin labeled MDM2 was eluted and detected by Western blotting withanti-MDM2 antibody. 1/20 of protein extracts was used for the detectionof GFP by Western blot to normalize for transfection efficiency.

MDM2 modulates the decay of TSG101. Earlier work has shown that bothTSG101 excess and deficiency can lead to abnormal cell growth (1) andthat the steady state level of TSG101 protein normally is regulatedwithin a narrow range by proteolysis (3). Just as TSG101 modulates theMDM2 level by negatively regulating its ubiquitination and decay, wefound that MDM2 has a parallel key role in the proteolysis of TSG101.This action of MDM2 was suggested initially by the observation that theintracellular concentration of endogenous TSG101 relative to α-tubulinwas markedly higher in p53^(−/−)/MDM2^(−/−) MEFs than in p53^(−/−) MEFcells, which are capable of producing MDM2 (FIG. 4 a). The correctnessof the notion that MDM2, which as already noted mediates the degradationof both itself and p53, also affects the decay of TSG101 was supportedby multiple lines of evidence. Firstly, the pulse-chase experiment seenin FIG. 4 b shows that the decay of endogenous TSG101 was accelerated inp53^(−/−) MEFs but was reduced in MEFs doubly mutated in p53 and MDM2.Secondly, SJSA-1 cells, which contain multiple copies of MDM2 as aresult of gene amplification (16), showed a major deficiency ofendogenous TSG101, as compared with cells (U205 and Saos-2) containing asingle chromosomal copy of MDM2 (FIG. 4 c). Thirdly, a dosage dependentdecrease of Flag-tagged TSG101 protein expressed from the CVM promoterwas observed in Saos-2 cells co-transfected with constructs that produceadventitious MDM2 (FIG. 4 d); because Saos-2 cells lack p53, thisexperiment also indicates that MDM2 mediated acceleration of TSG101decay does not require p53. Finally, while p53 overexpression, whichactivates endogenous MDM2 production (1, 16), was associated with adecrease in TSG101, overexpression of a mutant p53 protein that lacksthe ability to increase endogenous MDM2 had no effect on the TSG101level (FIG. 4 e).

That the effects of MDM2 on TSG101 degradation are, like those of p53,carried out by the 26S proteasome was shown by an experiment in whichaddition of the proteasome inhibitor, MG132, resulted in accumulation ofendogenous TSG101 (FIG. 4 f). Moreover, in the presence of MG132,overexpression of MDM2—which accumulated to >40× normal levels when theproteasome inhibitor was present—failed to accelerate decay of TSG101,confirming that MDM2-promoted decay of TSG101 requires proteasome action(FIG. 4 g).

FIG. 4. (a) Cellular TSG101 protein levels detected by Western blottingwith protein extracts (40 μg each) from mouse fibroblast NIH 3T3 cells,p53^(−/−) mouse embryo fibroblasts (MEF), and mouse embryo fibroblastsmutated in both p53 and MDM2. Relative densities of TSG101 protein bandswere calculated after normalization to cellular α-tubulin. (b) Cellcultures of NIH 3T3, p53^(−/−) MEF and p53^(−/−)/MDM2^(−/−) MEF werepulse labeled with ³⁵S-methionine for 1 hour, and chased for 0, 2, 4, 8and 12 hours. The ³⁵S-labeled TSG101 was immunoprecipitated byanti-TSG101 antibody, resolved in SDS gel, and visualized andquantitated by ImageQuant. (c) Vectors expressing Flag-tagged TSG101 andMDM2 were introduced into Saos-2 cells by co-transfection. Transfectingamounts of DNA are designated by + for 0.5 μg Flag-TSG101 expressionvector and + to ++++ for 1 to 4 μg MDM2 expression vector. (d)Combinations of vectors expressing MDM2, HA-tagged TSG101, and/or mutantor wild type p53 protein were introduced by transfection into Saos-2cells and protein extracts of transfectants were analyzed by Westernblotting using antibody as indicated. Where indicated, transfectantsreceived 1 μg plasmid DNA expressing HA-TSG101, 1 μg plasmid DNAexpressing MDM2, and p53-expressing constructs as follows: + and ++, 1μg and 2 μg respectively. The p53 mutation replaced the Arg at aa 175with His. (e) Saos-2 cell cultures were treated as indicated with MG132(2 μM) for 24 hours and cellular protein extracts were analyzed byWestern blotting with anti-TSG101 antibody. (f) Protein extracts fromSJSA-1, U2OS, and Saos-2 cells were analyzed by Western blot usingantibodies to MDM2, p53, TSG101 and α-tubulin. The ratio of MDM2/TSG101was determined after normalization with cellular α-tubulin. Theindicated constructs were introduced into Saos-2 cells by transfection(concentrations designated as in FIG. 4 d). Transfected cells werecultured in the absence or presence of MG132 for 24 hours and proteinextracts of cells were analyzed by Western blotting with anti-HA andanti-MDM2 antibody. The intensity of HA-TSG101 and MDM2 protein bands isindicated relative to cellular α-tubulin.

REFERENCES

-   1. Li, L. & Cohen, S. N. (1996) Cell 85, 319-29.-   2. Xie, W., Li, L. & Cohen, S. N. (1998) Proc Natl Acad Sci USA 95,    1595-600.-   3. Feng, G. H., Lih, C. J. & Cohen, S. N. (2000) Cancer Res 60,    1736-41.-   4. Gayther, S. A., Barski, P., Batley, S. J., Li, L., de Foy, K. A.,    Cohen, S. N., Ponder, B. A. & Caldas, C. (1997) Oncogene 15,    2119-26.-   5. Turpin, E., Dalle, B., de Roquancourt, A., Plassa, L. F., Marty,    M., Janin, A., Beuzard, Y. & de The, H. (1999) Oncogene 18, 7834-7.-   6. Lee, M. P. & Feinberg, A. P. (1997) Cancer Res 57, 3131-4.-   7. Sun, Z., Pan, J., Bubley, G. & Balk, S. P. (1997) Oncogene 15,    3121-5.-   8. Wagner, K. U., Dierisseau, P., Rucker, E. B., 3rd,    Robinson, G. W. & Hennighausen, L. (1998) Oncogene 17, 2761-70.-   9. Watanabe, M., Yanagi, Y., Masuhiro, Y., Yano, T., Yoshikawa, H.,    Yanagisawa, J. & Kato, S. (1998) Biochem Biophys Res Commun 245,    900-5.-   10. Sun, Z., Pan, J., Hope, W. X., Cohen, S. N. & Balk, S. P. (1999)    Cancer 86, 689-96.-   11. Hittelman, A. B., Burakov, D., Iniguez-Lluhi, J. A.,    Freedman, L. P. & Garabedian, M. J. (1999) Embo J 18, 5380-8.-   12. Koonin, E. V. & Abagyan, R. A. (1997) Nat Genet. 16, 330-1.-   13. Ponting, C. P., Cai, Y. D. & Bork, P. (1997) J Mol Med 75,    467-9.-   14. Hochstrasser, M. (2000) Science 289, 563-4.-   15. Levine, A. J. (1997) Cell 88, 323-31.-   16. Freedman, D. A. & Levine, A. J. (1999) Cancer Res 59, 1-7.-   17. Prives, C. (1998) Cell 95, 5-8.-   18. Oren, M. (1999) J Biol Chem 274, 36031-4.-   19. Lane, D. P. & Hall, P. A. (1997) Trends Biochem Sci 22, 372-4.-   20. Haupt, Y., Maya, R., Kazaz, A. & Oren, M. (1997) Nature 387,    296-9.-   21. Kubbutat, M. H., Jones, S. N. & Vousden, K. H. (1997) Nature    387, 299-303.-   22. Zhang, Y., Xiong, Y. & Yarbrough, W. G. (1998) Cell 92, 725-34.-   23. Sherr, C. J. & Weber, J. D. (2000) Curr Opin Genet Dev 10, 94-9.-   24. Buschmann, T., Fuchs, S. Y., Lee, C. G., Pan, Z. Q. &    Ronai, Z. (2000) Cell 101, 753-62.-   25. Baker, S. J., Markowitz, S., Fearon, E. R., Willson, J. K. &    Vogelstein, B. (1990) Science 249, 912-5.-   26. Levine, A. J., Wu, M. C., Chang, A., Silver, A., Attiyeh, E. F.,    Lin, J. & Epstein, C. B. (1995) Ann N Y Acad Sci 768, 111-28.-   27. Chen, J., Marechal, V. & Levine, A. J. (1993) Mol Cell Biol 13,    4107-14.-   28. Ward, C. L., Omura, S. & Kopito, R. R. (1995) Cell 83, 121-7.-   29. McMasters, K. M., Montes de Oca Luna, R., Pena, J. R. &    Lozano, G. (1996) Oncogene 13, 1731-6.-   30. Fiddler, T. A., Smith, L., Tapscott, S. J. &    Thayer, M. J. (1996) Mol Cell Biol 16, 5048-57.-   31. Honda, R. & Yasuda, H. (1999) Embo J 18, 22-7.-   32. Fang, S., Jensen, J. P., Ludwig, R. L., Vousden, K. H. &    Weissman, A. M. (2000) Biol Chem 275, 8945-51.-   33. Montes de Oca Luna, R., Wagner, D. S. & Lozano, G. (1995) Nature    378, 203-6.-   34. Jones, S. N., Roe, A. E., Donehower, L. A. & Bradley, A. (1995)    Nature 378, 206-8.-   35. Olson, D. C., Marechal, V., Momand, J., Chen, J., Romocki, C. &    Levine, A. J. (1993) Oncogene 8, 2353-60.-   36. Vousden, K. H. (2000) Cell 103, 691-4.-   37. Roth, J., Dobbelstein, M., Freedman, D. A., Shenk, T. &    Levine, A. J. (1998) Embo J 17, 554-64.-   38. Ruland, J., Sirard, C., Elia, A., MacPherson, D., Wakeham, A.,    Li, L., de la Pompa, J. L., Cohen, S. N. & Mak, T. W. (2001) Proc    Natl Acad Sci USA, 98 1859-64.-   39. Hsieh, J. K., Chan, F. S., O'Connor, D. J., Mittnacht, S.,    Zhong, S. & Lu, X. (1999) Mol Cell 3, 181-93.-   40. VanDemark et al., (2001), Cell 105, 711-720.-   41. Weissman (2001) Nature Reviews 2, 169-178.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention.

Various references are cited herein, each of which isincorporated-by-reference herein in its entirety for all purposes. Suchreferences include but are not limited to Li et al., (2001) Proc NatlAcad Sci USA 98, 1619-1624.

1-42. (canceled)
 43. A method of treating prostate cancer in a mammalcaused by abnormal function of TSG101 in cells of said mammal,comprising: administering a therapeutically effective amount of apharmaceutical composition comprising an antibody to TSG101 which bindsto an epitope in amino acid residues 1-250 of TSG101, and apharmaceutically acceptable excipient.
 44. The method of claim 43,wherein said mammal is a human.
 45. The method of claim 43, wherein saidantibody is a monoclonal antibody.